CN104075924B - Preparation and dielectrophoresis technology suitable for total protein of skeletal muscle of ruminants - Google Patents
Preparation and dielectrophoresis technology suitable for total protein of skeletal muscle of ruminants Download PDFInfo
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- CN104075924B CN104075924B CN201410336583.5A CN201410336583A CN104075924B CN 104075924 B CN104075924 B CN 104075924B CN 201410336583 A CN201410336583 A CN 201410336583A CN 104075924 B CN104075924 B CN 104075924B
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Abstract
The invention relates to an extracting and dielectrophoresis method for total protein of skeletal muscle of ruminants, and belongs to the field of biotechnologies. A dielectrophoresis technology is adopted for separation and analysis aiming at skeletal muscle of ruminants, in particular to sheep; experiments conducted on the skeletal muscle of sheep adopting the technical scheme acquire relatively excellent effect. Repeated experiments prove that the dielectrophoresis method is adopted to conduct separation on the skeletal muscle; acquired protein points after separation are multiple; repeatability is high; patterns are vivid; no horizontal and longitudinal line exists; the protein points are regular; repeatability is high; the dielectrophoresis method is a dielectrophoresis method suitable for analysis of total protein group of skeletal muscle of ruminants.
Description
Technical field
The present invention relates to a kind of holoprotein of ruminant skeletal muscle is prepared and bidirectional electrophoresis technique, this technology can directly be answered
Proteomics research for sheep and other ruminants.
Background technology
Due to the difference of the conditions such as growing environment, feed mode, the quality characteristic of mutton is had differences with pork, beef.
The crude protein content (12.8%-18.6%) of mutton is less than beef (16.2%-19.9%), higher than pork (13.5%-
16.4%).Crude fat content (16%-37%) is less than pork (25%-37%), higher than beef (11%-28%).Protein and
The difference of fat content, determines the technical conditions of ovine skeletal muscle dielectrophoresis.
The break-through point of the present invention is exactly the two-dimensional electrophoresis system setting up ovine skeletal muscle, can be to sheep bone bone using this technology
Flesh carries out proteomics research analysis, also may extend to the research of other ruminant meat quality characteristics.
Content of the invention
Present invention aim at providing a kind of holoprotein of ruminant skeletal muscle to extract and bidirectional electrophoresis technique, it is to be directed to
Ruminant albumen adopts bidirectional electrophoresis technique to prepare, through repeatedly testing, this technology proves that sample preparation is complete, reproducible, collection of illustrative plates
Clearly, it is a set of bidirectional electrophoresis technique being applied to ruminant skeletal muscle protein group analysis.
A kind of Two dimensional Electrophoresis of Proteins of ruminant skeletal muscle of the present invention, follow these steps to carry out：
Collection ruminant skeletal muscle, transports laboratory back, less than -80 DEG C save backup after liquid nitrogen flash freezer；
A) musculature preserving under the conditions of taking out -80 DEG C, weighs 1g and puts in mortar under freezing state, add appropriate liquid
Nitrogen is ground to powder；
B) dispense to centrifuge tube, add 10 times of volume lysates (7M urea, 2M thiocarbamide, 4%CHAPS, 40mM Tris,
40mM DTT, 1mmol/L protease inhibitors)；
C) it is aided with ultrasonication 10s, 4 DEG C of incubation 2h are centrifuged 1h in 4 DEG C of centrifuges of 10000 × g low temperature；
D) after skimming upper-layer fat layer, supernatant is dispensed；
E) quantification of protein：Bradford method, measures concentration and preserves after -80 DEG C；
F) first to isoelectric focusing electrophoresis：After measuring protein concentration, it is 1mg/ with hydrating fluid quantitative adjusting sample concentration
ML, Quantitative Western sample hydration liquid sample dissolution, draw 450 μ L sample solution and add in adhesive tape groove.Adhesive tape (pH3-10,
PH4-7 after) peelling off diaphragm, glue surface is lowered in adhesive tape groove it is ensured that covering one layer of mineral oil after not having bubble the voltolisation such as to prevent
Urea precipitation etc. when burnt.After adhesive tape takes passive aquation 12-16h, it is placed in Ettan IPGphor3 isoelectric focusing instrument, by setting
Two kinds of distinct program isoelectric focusing, control temperature be 20 DEG C.Every adhesive tape current limliting 50 μ A；
G) adhesive tape balance：Isoelectric focusing terminate rear adhesive tape first 10mL contain 1%DTT level pad I (6M urea,
75mM Tris-HCl, 29.3% glycerine, 2%SDS) middle balance 15min, uses the balance that 10mL contains 2.5% iodoacetamide afterwards instead
Balance 15min in buffer solution II (6M urea, 75mM Tris-HCl, 29.3% glycerine, 2%SDS)；
H) the second to SDS-PAGE electrophoresis：Adhesive tape is transferred to 12% separation gel upper end, uses 0.5% low melting-point agarose
Bind, carry out two to electrophoresis with maximum voltage (600V), electric current (400mA).Electrophoretic parameters are set to：Initial power 1W/ glue, fortune
After row 1h, power is carried to 8W/ glue, until terminating electrophoresis after Bromophenol Blue dye migration glue feather edge；
I) dye：Electrophoresis unloads offset plate after terminating, take out glue and dyeed.Respectively with improvement Coomassie brilliant G-250 dye
Color method, two methods of silver staining are dyeed.
J) decolour：G-250 dyeing terminates rear water elution and scans glue figure to background water white transparency, silver staining with 5% acetic acid
Wash glue.
It is characterised in that described gradual boosting, initial voltage is 50V to aforesaid method, then no less than 9h's
500V is slowly boosted in time.
It is characterised in that described gradual boosting, initial voltage is 50V to aforesaid method, keeps 6h, then rises again
It is depressed into 200V, then boost to 500V.
It is characterised in that described gradual boosting, initial voltage is 50V to aforesaid method, keeps 6h, then rises again
It is depressed into 200V, keep 1h, then boost to 500V.
Aforesaid method is it is characterised in that the solution of whole process is prepared and is both needed to use ultra-pure water.
Aforesaid method it is characterised in that the sample extraction stage need whole process to operate on ice or carry out suitable ice bath.
It is characterised in that the DTT needed for whole process is both needed to, existing use is existing to be added aforesaid method.
Aforesaid method is it is characterised in that described ruminant is sheep.
Brief description
Fig. 1 obtains glue figure for coomassie brilliant blue staining after direct boosting；
Fig. 2 is the glue figure after gradual boosting obtained by silver staining.
Specific embodiment
Embodiment 1
Using Neofuge15R type table-type high-speed refrigerated centrifuge and U.S.'s GE Two-dimensional Electrophoresis for Proteomics；
A) meat samples are gathered in slaughterhouse, liquid nitrogen flash freezer is transported back, -80 DEG C of preservations；
B) 1g meat samples are weighed after thawing, with adding liquid nitrogen to pulverize sample after removal lipid, manadesma；
C) 10 times of volume lysates are added, with cell crushing instrument ultrasonication 10s, 4 DEG C of incubation 2h are low after 10000 × g
It is centrifuged 1h in warm 4 DEG C of centrifuges；
D) after being centrifuged, upper strata lipid is removed in drift, draws appropriate clear liquid packing standby, if hyperliposis can Aspirate supernatant be carried out
Secondary centrifuging, to improve the recovery rate of protein；
E) quantification of protein：Protein content is measured using dying method with coomassie brilliant blue (Bradford method)；Using T6 system
Row ultraviolet-uisible spectrophotometer, with Coomassie brilliant G-250 dyeing, wavelength after quantification of protein normal gradients are diluted
595nm measures trap, does calibration curve with BSA, and measures protein content；
F) first to isoelectric focusing electrophoresis：After measuring protein concentration, it is 1mg/ with hydrating fluid quantitative adjusting sample concentration
ML, Quantitative Western sample hydration liquid sample dissolution, draw 500 μ L sample solution and add in adhesive tape groove.Adhesive tape (pH3-10,
PH4-7 after) peelling off diaphragm, glue surface is lowered in adhesive tape groove it is ensured that covering one layer of mineral oil after not having bubble the voltolisation such as to prevent
Urea precipitation etc. when burnt.After adhesive tape takes passive aquation 12-16h, it is placed in Ettan IPGphor3 isoelectric focusing instrument, by table 1
The program setting carries out isoelectric focusing, controls temperature to be 20 DEG C.
G) table 1IEF parameter setting：
| Program step | Pattern | Voltage/V | Time/V |
| 1 | Step | 250 | 1h |
| 2 | Step | 500 | 1h |
| 3 | Step | 1000 | 1h |
| 4 | Grad | 10000 | 2h |
| 5 | Step | 10000 | 80000Vh |
| 6 | Step | 500 | Random time |
H) adhesive tape balance：Isoelectric focusing terminate rear adhesive tape first 10mL contain 1%DTT level pad I (6M urea,
75mM Tris-HCl, 29.3% glycerine, 2%SDS) middle balance 15min, uses the balance that 10mL contains 2.5% iodoacetamide afterwards instead
Balance 15min in buffer solution II (6M urea, 75mM Tris-HCl, 29.3% glycerine, 2%SDS)；
I) the second to SDS-PAGE electrophoresis uses vertical electrophoresis, and sodium dodecyl sulfate gel liquid storage is 30% acryloyl
Amine, 0.8% methylene diacrylamide, 1.5mol/L pH8.8 Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tirs- alkali), 10% 12
Sodium alkyl sulfate (SDS), 10% ammonium persulfate (APS) and 1% tetramethylethylenediamine (TEMED) are prepared respectively, then mix
Close, water by volume: acrylamide: Tirs: SDS: APS: TEMED=5: 1.6: 2: 0.3: 0.05: 0.05: 0.002 configuration
12% concentration glue；
J) one terminate to electrophoresis after, carry out adhesive tape balance, adhesive tape is transferred to hectograph, enter second to electrophoresis.Under electrophoresis tank
Layer pours 1 × electrode buffer (1%SDS, 250mM Tris, 1.92M glycine) into, then hectograph is put into, and upper strata adds 2 × electricity
Pole buffer solution, carries out electrophoresis with maximum current and voltage, and 2w/ glue is adjusted to 8.5w/ glue after carrying out 1h terminates to electrophoresis, whole mistake
Journey, with recirculated water, environment temperature is controlled at 12 DEG C；
K) Coomassie brilliant G-250 according still further to improvement is dyeed (see Fig. 1).
Embodiment 2
Using Neofuge15R type table-type high-speed refrigerated centrifuge and U.S.'s GE Two-dimensional Electrophoresis for Proteomics；
A) meat samples are gathered in slaughterhouse, liquid nitrogen flash freezer is transported back, -80 DEG C of preservations；
B) 1g meat samples are weighed after thawing, with adding liquid nitrogen to pulverize sample after removal lipid, manadesma；
C) 10 times of volume lysates are added, with cell crushing instrument ultrasonication 10s, 4 DEG C of incubation 2h are low after 10000 × g
It is centrifuged 1h in warm 4 DEG C of centrifuges；
D) after being centrifuged, upper strata lipid is removed in drift, draws appropriate clear liquid packing standby, if hyperliposis can Aspirate supernatant be carried out
Secondary centrifuging, to improve the recovery rate of protein；
E) quantification of protein：Protein content is measured using dying method with coomassie brilliant blue (Bradford method)；Using T6 system
Row ultraviolet-uisible spectrophotometer, with Coomassie brilliant G-250 dyeing, wavelength after quantification of protein normal gradients are diluted
595nm measures trap, does calibration curve with BSA, and measures protein content；
F) first to isoelectric focusing electrophoresis：After measuring protein concentration, it is 1- with hydrating fluid quantitative adjusting sample concentration
1.5mg/mL, Quantitative Western sample hydration liquid sample dissolution, draw 500 μ L sample solution and add in adhesive tape groove.Adhesive tape (pH3-
10th, pH4-7) peel off diaphragm after glue surface be lowered in adhesive tape groove it is ensured that after there is no bubble cover one layer of mineral oil prevent
Urea precipitation etc. during electrofocusing.After adhesive tape takes passive aquation 12-16h, it is placed in Ettan IPGphor3 isoelectric focusing instrument, presses
The program isoelectric focusing of table 2, controls temperature to be 20 DEG C.
G) table 2IEF parameter setting：
| Program step | Pattern | Voltage/V | Time/V |
| 1 | Step | 50 | 6h |
| 2 | Step | 200 | 1h |
| 3 | Step | 500 | 2h |
| 4 | Grad | 1000 | 2h |
| 5 | Grad | 3000 | 3h |
| 6 | Grad | 5000 | 4h |
| 7 | Grad | 10000 | 4h |
| 8 | Step | 10000 | 50000Vh |
| 9 | Step | 500 | Random time |
H) adhesive tape balance：Isoelectric focusing terminate rear adhesive tape first 10mL contain 1%DTT level pad I (6M urea,
75mM Tris-HCl, 29.3% glycerine, 2%SDS) middle balance 15min, uses the balance that 10mL contains 2.5% iodoacetamide afterwards instead
Balance 15min in buffer solution II (6M urea, 75mM Tris-HCl, 29.3% glycerine, 2%SDS)；
I) the second to SDS-PAGE electrophoresis uses vertical electrophoresis, and sodium dodecyl sulfate gel liquid storage is 30% acryloyl
Amine, 0.8% methylene diacrylamide, 1.5mol/L pH8.8 Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tirs- alkali), 10% 12
Sodium alkyl sulfate (SDS), 10% ammonium persulfate (APS) and 1% tetramethylethylenediamine (TEMED) are prepared respectively, then mix
Close, water by volume: acrylamide: Tirs: SDS: APS: TEMED=5: 1.6: 2: 0.3: 0.05: 0.05: 0.002 configuration
12% concentration glue；
J) one terminate to electrophoresis after, carry out adhesive tape balance, adhesive tape is transferred to hectograph, enter second to electrophoresis.Under electrophoresis tank
Layer pours 1 × electrode buffer (1%SDS, 250mM Tris, 1.92M glycine) into, then hectograph is put into, and upper strata adds 2 × electricity
Pole buffer solution, carries out electrophoresis with maximum current and voltage, and 2w/ glue is adjusted to 8.5w/ glue after carrying out 1h terminates to electrophoresis, whole mistake
Journey, with recirculated water, environment temperature is controlled at 12 DEG C；
K) first use coomassie brilliant blue staining, carry out secondary dyeing with argentation：20min fixed by 50% methyl alcohol, 5% acetic acid
Afterwards, respectively wash 10min with 50% methyl alcohol, ultra-pure water, add 0.02% hypo solution sensitization 1min, pour precooling into
0.1% silver nitrate solution is incubated 20min in 4 DEG C, adds 0.04% formalin to be developed, whole process is tight after rapid washing
Lattice control time, is scanned (see Fig. 2) after decolouring.
In terms of the effect of Fig. 1, Fig. 2, because argentation sensitivity is high, obtained point is more, and puts more complete display,
It is analyzed by Image Master, the point on glue figure obtained by argentation is significantly more than Coomassie brilliant blue G250 method.
In isoelectric focusing program, table 1 is directly to boost, and table 2 is then gradual boosting.To different isoelectric focusing journeys after aquation
Sequence determines that the separation situation of protein is contrasted, and table 1 starts to boost for 250v, and another group is played slow boosting for 50v.250v group
Voltage change is very fast, and each voltage range boosting amplitude is big, and salinity is removed not exclusively, leads to Separation of Proteins situation poor；And from
50v plays slow boosting, arranges multistep low-voltage, has reached 9 hours to 500v, be easy to albumen and shift into IPG adhesive tape, contribute to
The removing of salt ion, desalination effect is obvious.It is slowly increased to the separation that high pressure also contributes to protein simultaneously.
Achieve successfully using the experiment of the technical program mutton, at present, through experiment repeatedly it was demonstrated that experimental repeatability is good,
Reliable results, can study for the ruminant proteins group such as sheep provides technical example.
Claims (7)
1. a kind of ruminant protein level of skeletal muscle bidirectional electrophoresis method is it is characterised in that follow these steps to carry out：
1) gather ruminant skeletal muscle sample, shipped back with liquid nitrogen container, save backup below -80 DEG C of laboratory；
2) weigh after 1g ruminant skeletal muscle thaws and put in mortar, add liquid nitrogen grinding, until being ground into powder, proceed to
The 15ml centrifuge tube of -20 DEG C of precooling；
3) add the protein cleavage liquid of 10 times of volume precoolings, after being sufficiently mixed, be aided with ultrasonication 10s；Wherein protein splits
Solution liquid is 7M urea, 2M thiocarbamide, 40mM Tris, mass volume ratio 4%3- [(3- cholesterol aminopropyl) dimethylamino] -1-
Propane sulfonic acid CHAPS, 65mM DTT, 1mM protease inhibitors and 2%IPG buffer；
4) 4 DEG C of incubation 2h are centrifuged 1h in 10000g, 4 DEG C of centrifuges of low temperature；
5) after being centrifuged, upper strata lipid is removed in drift, and it is standby to draw appropriate clear liquid packing, if hyperliposis can Aspirate supernatant carry out secondary
Centrifugation, to improve the recovery rate of protein；
6) quantification of protein：Its protein concentration is measured using Bradford method；
7) first to isoelectric focusing electrophoresis：By applied sample amount 450 μ g, loading volume 450 μ L, fixed measured protein sample is carried out
Dilution, loading hydrating fluid, with the lysate in step 3), after sample is added in retentive force IPG adhesive tape focusing disk, then is incited somebody to action
The IPG adhesive tape glue surface of pH 4-7, pH 3-10,24cm is lowered in disk, removes the bubble between glue surface and sample liquid, uses 2ml ore deposit
Thing oil/bar, covers adhesive tape, carries out isoelectric focusing electrophoresis；Gradual boosting is adopted in described isoelectric focusing electrophoresis；
8) adhesive tape balance：Retentive force IPG adhesive tape after focusing on well is balanced I, II, each equilibration time is 15min,
Level pad is formulated as follows：
Adhesive tape level pad mother liquor：6M urea, 2% (w/v) dodecyl sodium sulfate SDS, Tris-HCl of 75mM pH8.8,
29.3% 87% glycerine, solvent is water；
Adhesive tape level pad I：Every 10ml adhesive tape level pad mother liquor adds 0.1g DTT；
Adhesive tape level pad II：Every 10ml adhesive tape level pad mother liquor adds 0.25g iodoacetamide；
9) the second to SDS-PAGE electrophoresis：The adhesive tape having balanced is placed in the SDS-PAGE glue that gum concentration is mass volume ratio 12%
On, live top with mass volume ratio 0.5%, the low melting-point agarose sealing fluid-tight containing mass volume ratio 0.002% bromophenol blue,
Carry out electrophoresis by following parameter：Under 12 DEG C of constant temperature, under maximum current voltage, first use 1w/ glue, 1.5h；Use 8.5w/ glue again to electrophoresis
Terminate；
10) colouring method：Dyeed using Coomassie Brilliant Blue and argentation；
Described ruminant is sheep.
2. it is characterised in that described gradual boosting, initial voltage is 50V to method according to claim 1, then
500V is slowly boosted within the time no less than 9h.
3. it is characterised in that described gradual boosting, initial voltage is 50V to method according to claim 2, keeps
6h, then boosts to 200V again, then boosts to 500V.
4. it is characterised in that described gradual boosting, initial voltage is 50V to method according to claim 3, keeps
6h, then boosts to 200V again, keeps 1h, then boosts to 500V.
5. method according to claim 1 is it is characterised in that the solution of whole process is prepared and is both needed to use ultra-pure water.
6. method according to claim 1 it is characterised in that the sample extraction stage need whole process to operate on ice or fitted
Work as ice bath.
7. it is characterised in that the DTT needed for whole process is both needed to, existing use is existing to be added method according to claim 1.
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